1. Optimizing Purification and Quantitative Detection of Lysozyme and Avidin Tatiana Soboleva, Ryan Colakovic, Dr. Theresa Salerno (Faculty Mentor) tatiana.soboleva@mnsu.edu ryan.colakovic@mnsu.edu Department of Chemistry and Geology, Minnesota State University, Mankato, MN 56001 Abstract Background/Significance Table 1. Summary of Egg White Protein Characteristics Methodology Results Figure 1. The figure presents Western Blot analysis stages. Proteins are separated into individual chains on a 15% denaturing gel, and then are transferred to a nitrocellulose membrane. After blocking, the blot is exposed to specific primary and secondary antibodies and imaged. Conclusions References Acknowledgements Egg whites separatedpH adjustment with appropriate bufferHeat treatment at 65 o pH adjustment to 7.0 and centrifugeDialysisIon-exchange ChromatographyProtein quantitation Purification of Egg White Proteins Western Blot Analysis Gel Electrophoresis Separate the egg white proteins on a 15% denaturing Laemmli gel Electroblotting The proteins are transferred to a nitrocellulose membrane at 100V, 1.5 hrs. Immuno-detection The blot is exposed to anti-avidin or anti-lysozyme antibodies Secondary ab with IR labels is added 2o ab Hen egg white is one of the major raw materials used in food industry especially in foaming and gelling. Egg white consists of many different proteins that vary greatly in molecular weights and pI (isoelectric point) values. Two of its minor proteins, avidin and lysozyme have important commercial applications. In this research project, we tested the effect of pH and heat denaturation at 65 o on the purification and recovery of avidin and lysozyme proteins from egg white. Four different pH conditions were chosen; these included ascorbate buffers at pH values of 4 and 5 and phosphate buffers at pH values of 6 and 7. Following heat denaturation and dialysis, the minor proteins were purified by cation exchange chromatography. We also optimized a Western Blot method to quantitate differences in the recovery of the egg white proteins. In this method we were successfully detected small amounts of both proteins using IR detection. It was found that an ascorbate buffer with an initial pH of 5.0 gave the best recovery of both avidin and lysozyme proteins in the eluate 1 fraction. The pH values of 5 and 6 were the best for the removal of a major contaminant, ovotransferrin. These findings allow new insights into the future improvements in the purification of lysozyme and avidin, and have also provided methods that can be used in the laboratory project for Biochemistry 360 students. Experimental Design/ Objectives Egg white proteins differ considerably in their charge and mass properties. Lysozyme and avidin are minor protein species with important commercial applications. Both of these egg white proteins have very basic pI values and are commonly purified with cation exchange chromatography [3]. Improvements on this procedure might be obtained through the selective precipitation of contaminants with acidic pH buffers [4] and thermal denaturation [5]. Both lysozyme and avidin have been characterized using Western Blotting, but little has been done for avidin quantitative analysis. To optimize the purification of avidin and lysozyme by altering pH conditions during heat denaturation before cation exchange chromatography To develop a western blot procedure using fluorescence from near IR labeled antibodies for the quantitative detection of avidin and lysozyme and to use this to analyze E1 samples To use the developed Western Blot procedure to analyze the purity of the ion- exchange fractions at optimal pH conditions Both Eluate 1 and Eluate 2 (E1 and E2) fractions 50 mM Ascorbate pH 4.050 mM Ascorbate pH 5.050 mM Phosphate pH 6.050 mM Phosphate pH 7.0 Figure 2. Lysozyme Blot of E1 Figure 3. Avidin Blot of E1 pH= 4pH= 5pH= 6pH= 7 Lysozyme.6352.08.367.460 Ovotransferrin.660ND.338 pH= 4pH= 5pH= 6pH= 7 Avidin1.701.95.600.704 Ovotransferrin4.60.722.7393.54 Western Blot Analysis of E1 and E2 fractions Figure 4. Lysozyme Blot Figure 5. Avidin Blot pH 4.2 4, 65 o pH 4.2 4’, 65 o pH 4.2 8, 65 o pH 4.2 8, 65 o pH 4.8 4, 65 o Lysozyme (E1) ------------------------- Lysozyme (E2) 7.95 ---------- 115 3.63 --------- 101 4.56 ----------- 139 8.34 --------- 32.5^ 1.93 --------- 80.4 Ovalbumin Y (E2)ND Ovotransferrin (E2)ND.362ND [1] Gue Arin-Dubiard, C.; Pasco, M.; Molle, D.F. Proteomic Analysis of Hen Egg White. Rennes Cedex, France. 2013. [2] Li-Chan, E.; Nakai, S. Biochemical basis for the properties of egg white. Crit. ReV. Poult. Biol. 1989, 2, 21-58. [3]Thomas, B.R.; Vekilov, P.G.; Rosenberger, F. Heterogeneity Determination and Purification of Commercial Hen Egg- White Lysozyme. Acta Cryst. (1996).776-784. [4] STADELMAN, W.J.; COTTERILL, O.J. Egg science and technology. Westport: Avi Publishing, 1973. 314p. [5]Van der Plancken, A.Van Loey, ME.Hendrickx.Effect of heat-treatment one the physico- chemical properties of egg white proteins: A kinetic study. Newspaper of Food Engineering. 2006; 75: 316-326. Dr. Theresa SalernoDepartment of Chemistry and Geology Honors GrantBiochemistry 360 Students This document is available in alternative format to individuals with disabilities by calling Accessibility Resources at 507-389-2825 (V), 800-627-3529 or 711 (MRS/TTY). pH 4.2 4’, 65 o pH 4.2 4’, 65 o pH 4.2 8’, 65 o pH 4.2 8’, 65 o pH 4.8 4, 65 o Avidin (E1) ---------------------- Avidin (E2) 3.87 --------- 73.3 3.48 --------- 72.4 6.70 --------- 72.6 4.65 --------- 80.3 4.87 --------- 84.6 Ovalbumin Y (E2)7.4938.714.37.7834.3 Ovotransferrin(E2)ND2.57ND Table 4. Signal Intensities for Lysozyme BlotTable 5. Signal Intensities for Avidin Blot Western Blots were successfully obtained and detected low lysozyme and avidin levels in the eluate 1 (E1) fraction. Impure antibodies allowed for the detection of contaminating proteins. Signal intensities were determined for equal amounts of total protein. They suggest that lower pH conditions are optimal, but signals are low and duplicates were not run. The pH conditions at 5 and 6 were the best for the removal of ovotransferrin; the signal intensities were 5-6x lower. Optimization of the pH Conditions- Analysis by Western Blot The E2 fractions contained over 20x greater amounts of both avidin and lysozyme compared to the E1 fractions. The major contaminants were ovalbumin Y and ovotransferrin, but except for one sample ovotransferrin was not detected in the E2 fraction. Removal of ovalbumin Y was greatest at a pH of 4.2. Variability exist in student samples. Lysozyme was detected and quantitated with the Western Blot, and MW analysis gave a band of ~ 13kDa. Avidin was detected and quantitated on the Western Blot: its monomer molecular weight was ~15.9 kDa. Although the antibodies were not pure, this did not interfere with the analyses of the bands. Initial experiments suggested that lower pH optimizes yield but further experiments suggest that variability exists. The pH does affect the removal of contaminating samples. Further work should investigate the introduction of a normalizing control. Optimization of pH Conditions- Qualitative Observations Heat denatured samples at pH 7.0 and 6.0 were slightly cloudy whereas pH 5.0 had the most amount of precipitate formation. After pH adjustment the pH 4.0 sample did show precipitates. E1 E1 E1 E1 E1 E2 E2 E2 E2 E2 MW std. 4.2(4’) 4.2(8’) 4.2(4’) 4.8(4’) 4.2(8’) 4.2(4’) 4.2(8)’ 4.2(4’) 4.8(4’) 4.2(8’) E1 E1 E1 E1 E1 E2 E2 E2 E2 E2 MW std. 4.2(4’) 4.2(8’) 4.2(4’) 4.8(4’) 4.2(8’) 4.2(4’) 4.2(8)’ 4.2(4’) 4.8(4’) 4.2(8’) ^ bubble in blot MW pH=7 pH=6 pH=5 pH=4 Avidin pH=4 pH=5 pH=6 pH=7 MW